Acute lung injury is characterized by polymorphonuclear leukocyte (PMN) accumulation in the pulmonary microcirculation and transalveolar PMNmigration, and resultant injury of the microvessel-alveolar barriers. The mechanisms of migration of PMN across both pulmonary microvessel endothelial and alveolar epithelial barriers and the role of transalveolar PMN migration in the pathogenesis of acute lung injury remain unclear. In the proposed studies, we will direct the expression of the selective anti-adhesive beta2 CD11/CD18 integrin binding protein, Neutrophil Inhibitory Factor (NIF), in a site- and inflammation- specific manner. NIF will be expressed in either pulmonary microvascular endothelium or type II alveolar epithelial cells using inducible cell-specific promoters. Constructs of NIF cDNA driven by the inducible endothelial cell-or type II alveolar epithelial cell-specific promoters (i.e., E-selectin and surfactant protein C promoters, respectively) will be introduced in mice to express NIF at endothelial and epithelial sites. Mice will be challenged with TNFalpha, or with gram negative or gram positive bacteria, E. coli or S. pneumoniae, administered intra- peritioneally or directly into airway to simulate systemic or lung-localized infection. We will determine the effects of site- specific PMN beta2 integrin blockade in either microcirculation or alveolar space on the migration of PMN across microvessel- alveolar epithelial barriers and in the injury of these barriers. We will address the role of beta2 integrins in directing PMN traffic across the pulmonary microvessel endothelial and alveolar epithelial barriers and mechanism of injury of these barriers using a strategy based on cell-selective and inducible expression of beta2 integrin antagonist. In separate studies, we will determine using gene knockout mouse models the contributions of L-selectin (another PMN adhesion molecule involved in PMN migration) as well as ICAM-1 (a PMN beta2 integrin counter- receptor in endothelial and alveolar epithelial cells) and E- selectin (an endothelial cell-specific adhesion molecule) in the mechanisms of alveolar PMN migration and injury of microvessel and alveolar epithelial barriers. With the completion of these studies, new information will be obtained on the role of adhesion molecules in the mechanisms of PMN migration into the airspace and novel strategies will be developed to prevent acute lung injury based on the conditional and site-specific expression of a beta2 integrin antagonist.